PROJECT SUMMARY Francisella tularensis (Ft) causes tularemia, a serious and potentially fatal disease. Because Ft has an extraordinarily high infectivity, causes high morbidity and mortality, is relatively easily cultured and dispersed, and has previously been weaponized, it is classified as a Category A potential agent of bioterrorism. A safer and more potent vaccine against aerosolized Ft than the current unlicensed, toxic, and insufficiently effective vaccine (LVS) is needed, and that is the goal of this application. Our strategy is to utilize a live attenuated recombinant homologous vector - an attenuated form of the LVS vaccine (already tested in humans) - to overexpress highly immunoprotective Ft proteins such as IglC, previously demonstrated in this laboratory to enhance protective immunity against aerosolized Ft. The recombinant LVS (rLVS) vaccine will be safer than LVS because the proposed vector has been demonstrated to be >10,000 more attenuated than LVS and to induce strong cell-mediated and humoral immune responses; the vaccine will be more potent because it will overexpress highly immunoprotective Ft proteins. This strategy mimics that used successfully in this laboratory to develop the first vaccines against tuberculosis that are safer and more potent than the current BCG vaccine;one of these vaccines has already demonstrated safety and enhanced immunogenicity in human trials. Our primary goal is a single vaccine against tularemia that is safer and more potent than LVS, but as an alternative, we shall optimize a heterologous booster vaccine using an improved attenuated Listeria monocytogenes (Lm) vector to express the same immunogenic Ft protein(s) as the rLVS vector. The Lm vaccine vector has an excellent safety (it has been safely administered to humans), efficacy, and cost profile. We shall determine the efficacy of a heterologous prime-boost vaccination strategy using the rLVS vaccine as a prime and an improved rLm vaccine as a boost. Our previous studies have demonstrated moderate efficacy of the rLm vaccine by itself against both lethal i.n. LVS challenge and aerosol Ft SchuS4 challenge and great promise as a heterologous booster vaccine;moreover, the rLm vaccine is effective after intradermal administration, a safe and convenient route. We propose to build upon our initial success by developing new live attenuated recombinant vaccines and testing them systematically for safety, immunogenicity, and short-term and long-term efficacy against lethal Ft challenge in mouse models. By the completion of this project, we anticipate having a vaccine that is safer and more potent than LVS and suitable for testing in humans. While the focus of our grant is on a tularemia vaccine, our approach is applicable to vaccines against intracellular pathogens in general. Thus, lessons learned and strategies developed during the development of a successful tularemia vaccine are likely to be broadly applicable.